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Degradability and biocompatibility of magnesium-MAO: The consistency and contradiction between in-vitro and in-vivo outcomes
⁎Corresponding authors. qh197826@163.com (Hui Qin), hlc@mail.sic.ac.cn (Huiliang Cao), anzhiquan@126.com (Zhiquan An)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Abstract
Magnesium-based metals are promising for biomedical use for its biodegradation characteristic. However, its rapid degradation in physiological environment impedes its application. Oxides coating is a widely-used form of surface modification to improve the corrosion resistance of biomaterials. In this study, we systematically compared two oxides coatings, steam oxidation (SO) and micro-arc oxidation (MAO), in controlling degradation and improving the biocompatibility of pure magnesium in vitro and in vivo. The addition of both coatings was found to reduce the degradation rate of magnesium in vitro at the early stage, but the corrosion behavior became severe with longer immersion time in all the groups. Unfortunately, all the coated and uncoated materials were found undermining the adhesion and proliferation of MC3T3-E1 cells in vitro, even at the first day of culture. In terms of in-vivo circumstance, the Mg-MAO group showed satisfying biocompatibility compared with pure Mg and Mg-SO, as is evidenced by radiographic results and histological analysis. Little corrosion was found, and more newly formed bone was observed in the animal model. These data suggest that the characteristic of in-vivo circumstance have considerable impacts on the degradation and bone integration process.
Keywords
Micro-arc oxidation
Magnesium
Biocompatibility
Surface modification
1 Introduction
The most commonly used biomaterials for bone defects are stainless steel, pure titanium and its alloys (Wieding et al., 2015), and cobalt–chromium alloys (Shah et al., 2016), which are all non-biodegradable. However, many problems associated with these permanent metallic materials may arise after the bone healing. With the long-term irritation, inflammatory reactions may occur as the metallic implant is regarded as a foreign body for human tissues (Cipriano et al., 2017; Liu et al., 2016; Tsaryk et al., 2013). If this is the case, these side effects may subsequently require a second surgery for implant removal after the healing of the injury. However, repeated surgery not only increases the risk associated with additional surgical procedures, but also results in increased health care costs and longer hospitalization (Olsen et al., 2017).
Biodegradable magnesium-based metals are currently breaking the paradigm in developing only highly corrosion-resistant metals for implantable biomedical devices, such as stents, plates, and screws (Chaya et al., 2015; Zhao et al., 2016). Magnesium is the most commonly used metallic implant amongst all the biodegradable metallic materials, whereas its toxicity resulted by rapid degradation and aggressive release of hydrogen gas in physiological environment impedes its broad applications (Draxler et al., 2017; Martinez Sanchez et al., 2017). Hence, the vulnerable corrosion resistance of magnesium must be improved to utilize magnesium-based materials for biomedical application (Khan et al., 2016).
The degradation characteristic of pure magnesium can be modified by alloying (Fazel Anvari-Yazdi et al., 2016; Gil-Santos et al., 2017; Peng et al., 2016) or various surface treatments (Kaabi Falahieh Asl et al., 2016; Yazdimamaghani et al., 2015; Zhao et al., 2016). Accordingly, Mg-Zn alloy, Mg-Ca alloy, Mg-Mn-Zn alloy, Mg-Zn-Zr alloy, and Mg-rare earth alloy have been developed for biomedical implantation. Our research group have also successfully fabricated Mg-Nd-Zn-Zr alloy and found that this magnesium alloy effectively enhances the corrosion resistance and biocompatibility of magnesium (Qin et al., 2015). The elements used in magnesium alloys should be selected and evaluated carefully considering their cytocompatibility and mechanical properties during degradation (Cheng et al., 2016; Mochizuki et al., 2016). Apart from alloying, surface modifications have also been widely investigated and oxides coating has shown an increase effects in improving the corrosion resistance property. Compared with alloying, metal oxides have the advantages of stability, intrinsic semiconductivity, and composition of non-toxic and earth-abundant elements (Tubtimtae et al., 2013). Steam oxidation (SO) treatment is a common oxidization method and could rapidly grow crystalline films on magnesium surface which has anti-corrosion effects (Hoye et al., 2015). Micro-arc oxidation (MAO), an improved method to form oxides coating (Dou et al., 2017), has been widely utilized on magnesium substrates in industrial applications to fabricate protective coatings (Li et al., 2015; Razavi et al., 2015). However, the porous structure of the coating surface would allow corrosion electrolytes to permeate into the coating, which could reduce its corrosion resistance in vitro (Razavi et al., 2014; Shangguan et al., 2016). To further improve the corrosion resistance and biocompatibility, duplex techniques like poly-L-lactic acid (PLLA) were used to seal the porous surface of MAO coating (Lu et al., 2011). However, the degradable characteristic of magnesium substrate would be impeded by sealed coating.
At in-vivo environment, there are much more complicated circumstances than the in-vitro conditions. The corrosion resistance property and biocompatibility of the substrate should be different between in-vitro and in-vivo environment. The objective of this paper is to systematically study the effects of oxidized coating in modifying the degradation property of pure magnesium under in-vitro and in-vivo conditions and to evaluate whether the MAO process is adequate to improve the biocompatibility of pure magnesium.
2 Material and methods
2.1 Materials preparation
The 99.9% pure magnesium (a gift from Southeast University, Nanjing, China) was cut into 10 mm × 10 mm × 2 mm, 20 mm × 20 mm × 2 mm, and 2 mm × 2 mm × 10 mm. Then, some of the samples were treated by micro-arc oxidation by a modified procedure (denoted as Mg-MAO) previously described (Yao et al., 2009). The MAO electrolyte used in the present study is consisted of sodium silicate (12 g/L), sodium hexametaphosphate (2 g/L) and distilled water (remaining). The MAO duration is 2 min and the working voltage is 300 V. And some of the samples were treated by steam oxidation in an autoclave at 121 °C for 40 min (denoted as Mg-SO). The remaining pure magnesium samples were studied as control group, denoted as Mg.
2.2 Material characterizations
Surface morphologies and profiles of the concerned elements were analyzed by an energy dispersive spectroscopy (EDS) coupled scanning electron microscopy (HITACHI S-3400, Japan). The chemical states of the elements on the sample surface were evaluated by X-ray photoelectron spectroscopy (XPS, PHI 5802, Physical Electronics Inc., Eden Prairie, MN).
2.3 Potentiodynamic polarizations
Potentiodynamic polarization tests were performed on the samples in a physiological saline solution (0.9% NaCl at a pH of 7) using a electrochemical workstation (CHI760C, Shanghai, China). The measurement was conducted at room temperature using a conventional three-electrode electrochemical cell with a graphite rod as the counter electrode, a saturated calomel electrode (SCE) as the reference electrode, and the sample with a 0.785 cm2 exposed area as the working electrode. Prior to the polarization test, the samples were immersed in the saline solution for 15 min to establish a relatively steady open circuit potential, and the tests were conducted at a scanning rate of 10 mV/min.
2.4 Immersion tests
In order to monitor the degradation behavior of the coated and uncoated magnesium samples, immersion test was designed and carried out at different time points. Six parallel ones of each group were individually immersed into a sealable centrifugal tube containing 40 mL physiological saline solution and then incubated at 37 °C for as long as 30 days. The morphologies of different samples after immersion were observed at 1, 7 and 30 days under scanning electron microscope. The concentration of released magnesium ions was measured using inductively-coupled plasma mass spectrometry (ICPMS) (Optical Emission Spectrometer, Perkin Elmer, Optima 2100DV). In addition, the pH changes of the solutions were also measured.
2.5 In-vitro cytocompatibility
To evaluate the cytocompatibility of the materials, standard cell culture was applied on the surfaces of the coated and uncoated samples. The MC3T3-E1 cells (Cells Resource Center of Shanghai Institute for Biological Science, Shanghai, China) were incubated at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. The α-Modified Eagle’s Medium (α-MEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) was used as culture media. The cells of 5 to 7 generations were used in this study. Samples sterilized by ultraviolet radiation for 10 h were put into 24-well culture plates (Costar, USA). Cell suspension (1 mL) consisting of 1 × 105 cells/mL was added to each well. Cells were incubated at 37 °C in a humidified atmosphere of 5% CO2 and 95% air for 4 h to attach on the sample surfaces. The samples were then transferred to another 24-well culture plates and 1 mL culture medium was added into each well for further culture. After 1 day of culture, the materials were taken out and rinsed by phosphate buffered saline solution (PBS, pH = 7.4) twice to remove unattached cells. All samples were fixed by 2.5% glutaraldehyde solution for 4 h at 4 °C. Then the samples were dehydrated in graded ethanol series [30, 50, 70, 80, 90 (%, v/v)] for 10 min, respectively, with final dehydration performed in absolute ethanol twice for 10 min. The samples were put into the refrigerator at -80 °C for 2 h, and then vacuumed in freezer dryer. The cell morphology was observed by SEM.
2.6 In-vivo studies
2.6.1 Surgical procedure
All animal procedures described in this article were performed according to established guidelines for animal care and have been approved by the Animal Care and Use Committee of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital.
Male Sprague-Dawley Rats with a body weight of 150–170 g were used in this experiment. The rats were randomly divided into three groups (6 parallel rats in each group), and the substrate of Mg, Mg-SO or Mg-MAO was implanted into the femur of each rat. The rats were anaesthetized with 10% chloral hydrate solution (3 mL/kg) by intraperitoneal administration. Their legs for the operation were shaved. And then, a hole (approximately 2.5 mm in diameter) was drilled between the condyles of the distal femur through a minimally invasive approach. Subsequently, the substrate was implanted into the drilled hole. The wound was then sutured layer by layer. After the operation, all rats received intraperitoneal injections of antibiotics for 3 days. The rats were allowed to move in their cages freely with unrestricted weight bearing.
2.6.2 Radiographic evaluations
At each particular time point (1 day, 1 and 3 months after operation), X-ray radiography (digital DIAGNOST, Philips Medical) was conducted at the implantation site so as to monitor the sample degradation and bone healing process.
2.6.3 Micro-CT analysis
In order to further monitor the corrosion behavior of the samples, at 4, 8 and 12 weeks after the operation, the rats were scanned in a micro-computed tomography device after general anaesthetization. All the scans were performed by a SkyScan 1176 compact X-ray MicroCT scanner (Bruker, Belgium) with beam set at 90 kV and 270 μA. The effective pixel size was 18 μm. After scanning, 2D planes were reconstructed by the SkyScan Dataviewer (Bruker, Belgium). The residual implant volume and new bone growth were quantified by SkyScan CTAn program (Bruker, Belgium).
2.6.4 Magnesium ion levels in blood
Blood was collected prior to the operation, and 1 week, 4 weeks, 8 weeks after the operation to test the magnesium ion concentrations in blood. Blood was centrifuged at 1300g for 15 min at room temperature and the sera were collected and stored at 4 °C before analysis. Prior to analysis, the sera were diluted for 10 times in double distilled water. The magnesium ion concentration was determined by inductively-coupled plasma mass spectrometry (ICPMS) (Optical Emission Spectrometer, Perkin Elmer, Optima 2100DV).
2.6.5 Sequential fluorescent labeling assays
Fluorescent labeling was applied to evaluate the new bone formation at the different time. The rats were intraperitoneally administered with 20 mg/kg calcein (CA, Sigma, USA), 30 mg/kg alizarin red (AL, Sigma, USA) and 25 mg/kg tetracycline (TE, Sigma, USA) at 2, 6, 10 weeks after the operation, respectively.
2.6.6 Histological observations
The bone samples containing the implants were harvested and fixed in 4% paraformaldehyde. Subsequently, a step of standard tissue processing was conducted to transfer the samples from the aqueous stage to the organic stage. Dehydration was performed using ascending grades of ethanol (70%, 75%, 80%, 85%, 90%, 95% and then twice 100%). The samples were immersed in each solution for 1 day. The samples were then immersed in xylene for 4 h. Finally, all the samples were embedded in methyl-methacrylate. The embedded samples were cut into sections with a thickness of 250 μm and then ground and polished to a thickness of 30 μm. The sections were observed using confocal laser scanning microscope (Zeiss LSM710, Germany) for fluorescent labeling. Subsequently, the sectioned samples were treated with Van Gieson staining. The morphological analyses were performed under a light microscope to observe any material corrosion and bone formation.
2.7 Statistical analysis
Statistically significant differences between the various groups were measured using two-way ANOVA. The statistical analysis was carried out using IBM SPSS Statistics Version 19.0 (SPSS Inc., Chicago, Illinois, USA). The data are expressed as the mean ± standard deviation (SD).
3 Results
3.1 Surface characterization
Fig. 1 shows the surface morphologies of Mg, Mg-SO, and Mg-MAO under SEM. Accordingly, it can be concluded that a thin film was formed on the Mg-SO and Mg-MAO group. The cross-section of the films was analyzed by energy dispersive spectroscopy (EDS) coupled SEM, and the results (Figs. S1, S2, and S3) show that the surface layer obtained by steam oxidation (SO) is about 1.5 µm (Fig. S2) while that produced by micro-arc oxidation (MAO) is about 2.0 µm (Fig. S3). The MAO coating is relatively dense because we used a short MAO duration (2min) and low working voltage (300 V) in the present study. The X-ray photoelectron spectroscopy (XPS) full spectra obtained from the Mg, Mg-SO and Mg-MAO surfaces are presented in Fig. 2. Although oxygen (O) presented in all the three groups, its intensity is different. The intensity of oxygen in Mg-SO and Mg-MAO groups is apparently higher than that in the Mg group, indicating that both steam oxidation and micro-arc oxidation can produce an oxidic layer on the magnesium substrate. Moreover, a small amount of sodium (Na) and phosphorus (P) was presented on Mg-MAO group resulted by the electrolyte components. The XPS high-resolution spectra of Mg 2p and O1s (Fig. S4) demonstrated that spontaneously formed magnesium oxide is presented on the Mg group, the surface layer of the Mg-SO group is composed of magnesium hydrate, and the surface layer of the Mg-MAO group consists of magnesium oxide and trace concentration of other oxides (they may be carbon, silicon, phosphorus contained, and it is hard to confirm exactly at present).
Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.arabjc.2018.07.010.
Fig. 1 shows the surface morphologies of Mg, Mg-SO, and Mg-MAO under SEM. Accordingly, it can be concluded that a thin film was formed on the Mg-SO and Mg-MAO group. The cross-section of the films was analyzed by energy dispersive spectroscopy (EDS) coupled SEM, and the results (Figs. S1, S2, and S3) show that the surface layer obtained by steam oxidation (SO) is about 1.5 µm (Fig. S2) while that produced by micro-arc oxidation (MAO) is about 2.0 µm (Fig. S3). The MAO coating is relatively dense because we used a short MAO duration (2min) and low working voltage (300 V) in the present study. The X-ray photoelectron spectroscopy (XPS) full spectra obtained from the Mg, Mg-SO and Mg-MAO surfaces are presented in Fig. 2. Although oxygen (O) presented in all the three groups, its intensity is different. The intensity of oxygen in Mg-SO and Mg-MAO groups is apparently higher than that in the Mg group, indicating that both steam oxidation and micro-arc oxidation can produce an oxidic layer on the magnesium substrate. Moreover, a small amount of sodium (Na) and phosphorus (P) was presented on Mg-MAO group resulted by the electrolyte components. The XPS high-resolution spectra of Mg 2p and O1s (Fig. S4) demonstrated that spontaneously formed magnesium oxide is presented on the Mg group, the surface layer of the Mg-SO group is composed of magnesium hydrate, and the surface layer of the Mg-MAO group consists of magnesium oxide and trace concentration of other oxides (they may be carbon, silicon, phosphorus contained, and it is hard to confirm exactly at present).
Supporting material
Supporting material3.2 Potentiodynamic polarization
Fig. 3 presents the potentiodynamic polarization curves of Mg, Mg-SO and Mg-MAO in physiological saline solution, and the corresponding photos taken at the surface of the samples after the tests. The corrosion potential (Icor), corrosion potential (Ecor), and Tafel slopes (the cathodic slope denoted as Bc, anodic slope denoted as Ba) of the samples were listed in Table 1. The results demonstrated that both SO and MAO treatment can reduce the corrosion of the magnesium substrate, and the Mg-MAO group was better than Mg-SO group.
| Mg | Mg-SO | Mg-MAO | |
|---|---|---|---|
| Icor (A/cm2) | 4.10E−5 | 1.81E−6 | 3.27E−7 |
| Ecor (mV) vs. SCE | −1547 | −1394 | −953 |
| Tafel slopes (V.dec-1) | Bc = 6.269 Ba = 12.684 |
Bc = 4.261 Ba = 20.774 |
Bc = 3.168 Ba = 7.120 |
3.3 Immersion tests
The degradation property of Mg, Mg-SO and Mg-MAO was further evaluated by immersing them in physiological saline solution for a maximum of 30 days. The surface morphologies of the samples after immersion were observed by SEM. The results (Fig. 4) showed that severe localized corrosion happened on Mg specimen. Micro-cracks were formed on the material surfaces and the corrosion products increased over time (Fig. 4A, D and G). In the case of the Mg-SO group, there were also severe corrosion cracks formed on the samples (Fig. 4B, E and H), and increasing spherical product can be observed on the surface. For the Mg-MAO group, there was little change at an early stage. However, most micro-pores of the MAO coating were filled with corrosion products, and micro-cracks were observed after 7 days’ immersion. The corrosion was even severer after 30 days (Fig. 4C, F and I).
The amount of magnesium ion released into the physiological saline solution was measured using the inductively-coupled plasma mass spectrometry (ICPMS), and the results were shown in Fig. 5. It was found that the magnesium concentration of the Mg group increased from approximately 344 ppm after 1 d of immersion, to 547 ppm after 30 days of immersion, whereas the magnesium concentration of the Mg-SO and Mg-MAO samples had a more gentle increase. The magnesium ion concentrations detected between 1 d and 30 d of the immersion test in Mg-SO and Mg-MAO groups ranged between 193 ppm and 513 ppm and from 101 ppm to 467 ppm, respectively.
The pH value of the solutions increased over time. The pH range of Mg-SO and Mg-MAO groups were found to be about from 8.5 to 9.6 and 7.8 to 9.0, respectively. However, the pH value of the Mg group was apparently higher ranging approximately from 9.0 to 10.3 (see Fig. 6).
3.4 In-vitro cytocompatibility
Fig. 7 shows the surface morphologies of the samples, on which MC3T3-E1 cells were cultured for 24 h. No intact cells were found on all the samples, indicating that both SO and MAO treatments were inadequate to improve the in-vitro cytocompatibility of magnesium, though they were effective in reducing the degradation of magnesium.
3.5 In-vivo compatibility
3.5.1 Radiographic evaluations
Fig. 8 shows the radiographs of the rat femur at 4, 8 and 12 weeks after the operation. Large amounts of the gas bubble were observed in Mg and Mg-SO groups around the implantation site at 4 and 8 weeks. Bone absorption can also be seen around the Mg and Mg-SO samples. At 12 weeks, the gas bubble was mostly absorbed in Mg-SO group, while large gas cavity could still be observed in the femur of Mg group. In contrast, only a little bubble formation was observed around the Mg-MAO implants during the implanting period. Slight bone absorption was observed at 4 weeks, and the bone defect was found healing at 8 weeks.
3.5.2 Micro-CT analysis
The in-vivo morphology of the implant in the rat femur was measured using Micro-CT. Fig. 9 shows the cross sections of the corroded implants and the surrounding biological response within 12 weeks. The Mg group underwent severe corrosion at the surface and generated a large amount of gas after implantation. A small quantity of new bone was formed around the implant. The corrosion of Mg-SO group was slighter than the Mg group, and more new bone was formed around the implants. In the Mg-MAO group, there was not apparent gas around the implants. Only a little corrosion could be seen on the surface. High-density bone layers can be observed around the implant. Table 2 shows the volume of newly formed bone around the implants and the volume reduction of the implants. The Mg implant showed the least volume of new bone formation of 0.56 mm3 and the largest implant volume reduction of 14.36% after 12 weeks of implantation. For the Mg-SO group, a larger amount of new bone growth (1.52 mm3) and less implant volume reduction was presented (7.17%). The Mg-MAO group showed the smallest implant volume reduction (2.38%) and largest new bone formation (4.72 mm3) than the other two groups.
| New bone volume (mm3) | Initial implant volume (mm3) | Final implant volume (mm3) | Implant volume change (%) | |
|---|---|---|---|---|
| Mg | 0.56 | 28.35 | 24.28 | −14.36% |
| Mg-SO | 1.52 | 28.35 | 26.32 | −7.17% |
| Mg-MAO | 4.72 | 28.35 | 27.68 | −2.38% |
3.5.3 Serum magnesium levels
Fig. 10 shows the changes of serum magnesium ion concentration in the three groups after the operation. The serum magnesium levels of all the rats were observed to fluctuate from approximately 2.2 ppm to 3.5 ppm.
3.5.4 Bone formation
Fig. 11 shows the image of fluorescent quantification, which demonstrated the bone deposition and mineralization level at different time. At 2 weeks after operation, the percentage of CA labeling (green) in Mg-SO group was 1.56 ± 0.17%, which was more than that in Mg Group (1.26 ± 0.18%), but less than that in Mg-MAO group (3.76 ± 0.26%) (Fig. 11A1, B1, C1). There were significant differences among the three groups (p < 0.05, Fig. 12). At 6 weeks, the percentage of AL labeling (red) was 1.13 ± 0.14%, 1.58 ± 0.13%, and 2.06 ± 0.15%, in the Mg, Mg-SO and Mg-MAO groups, respectively (Fig. 11A2, B2, and C2). The differences were significant among the three groups (Fig. 12). At 10 weeks, the percentage of TE labeling (yellow) was 0.85 ± 0.06%, 1.01 ± 0.09% and 1.19 ± 0.06%, respectively (Fig. 11A3, B3, C3), with statistically significant differences among the three groups (Fig. 12). Taken together, the data indicates that, Mg-MAO presented better effects on promoting the new bone formation and mineralization than Mg and Mg-SO, especially at an early stage. The Mg-SO group was more effective than the Mg group in promoting new bone formation and mineralization, but less than the Mg-MAO group.
3.5.5 Histological evaluations
Fig. 13 shows the interface between mineralized bone tissue and the implants after 12 weeks of implantation. All the implanted samples presented direct contact with the newly formed bone. Evident more bone was formed around the MAO-coated group in comparison to the uncoated and the SO-coated group.
4 Discussion
In this study, pure magnesium (Mg) substrates were treated by steam oxidation (SO) and micro-arc oxidation (MAO) to produce different oxide coatings. The degradation of Mg substrates in physiological saline solution was reduced with these coatings. However, the in-vitro cytocompatibility of these two coatings to MC3T3-E1 cells was only limitedly improved. Even so, in-vivo experiments demonstrated that both SO and MAO coated implants could apparently improve the bone integration, and the Mg-MAO group was even better.
Magnesium and its alloys, as biodegradable materials, has attracted much attention, whereas the degradation of magnesium releases a large amount of Mg2+, and causes local alkalization and hydrogen accumulation, which may be toxic to mammalian cells and impede tissue repair. Micro-arc oxidation could fabricate an oxide layer on magnesium substrates. However, it was considered that this technique is inadequate to improve the cytocompatibility of magnesium because cells can hardly accommodate themselves on the material (Wei et al., 2015).
In this study, the SO and MAO coated magnesium did not perform well enough in corrosion resistance and cytocompatibility under in-vitro condition. The MAO coated substrate exhibited best anti-corrosion property among the three groups at the early immersion stage and. However, small cracks of corrosion could also be observed in the Mg-MAO group after immersed in the physical saline solution for 7 days long. The cytocompatibility is highly influenced by pH changes and hydrogen evolution as the cells are sensitive to these parameters. In terms of magnesium, the rapid degradation leaded to a local increase in pH value and Mg2+ ion concentration, which is the main obstacle for cell adhesion. Moreover, the cells attachment on the surface could be impeded by the released hydrogen. On the contrary, the pH values of the solution in Mg-SO and Mg-MAO groups increased more slightly as the coatings acted as a protecting layer. However, the SEM result of cell adhesion on SO and MAO coated sample surfaces still demonstrated an inhibited outcome, even at the first day of the culture.
In terms of the in-vivo experiment, the number of gas bubbles and bone formation around the implanted samples were observed by radiographic assay. Gas cavity generated around the implants in vivo due to excess gas evolution caused by the implant degradation. The bone absorption may be caused by locally increased pH value and Mg2+ ion concentration. Interestingly, the Mg-MAO group showed a different result. The MAO coating could effectively reduce the gas evolution in vivo. The bone absorption was also slighter around the MAO-coated implants. In Micro-CT test, slightest corrosion occurred on the surface of MAO coating. Even after implantation of 12 weeks, the MAO coated samples almost maintained their original shape with newly formed bone around them. The outcomes of histological analysis were corresponded to the Micro-CT analysis. No severe inflammation and necrosis was observed in the surrounding tissues in the Mg-MAO group. There were no significant changes in serum magnesium value after the operation for all the groups as the excess magnesium ion will be excreted by the kidney (Kim et al., 2014; Wang et al., 2016). The values of serum magnesium ion in different group at different time were all below 3.5 ppm and not beyond the normal range of physiological magnesium levels (Vormann, 2003; Wlaz et al., 2016). The results indicated that there should be no toxic effects occurring in vivo for MAO-coated implants.
These results solidly demonstrated that the bone integrating effect of materials in vivo could be satisfying even the in-vitro cytocompatibility was not good enough. In fact, the low cytocompatibility of SO and MAO coating in vitro could be due to the gradual corrosion of the material. Even though MAO could enhance the corrosion resistance of substrate in vitro at the early stage, the Mg2+ concentration and pH value would still reach a toxic level for cells when the materials were continuously immersed. In Contrary, the materials implanted into living organism will simultaneously interact with body fluids, tissues and cells (Ma et al., 2007; Wong et al., 2013). The initial response starts with protein adsorption under the biological system (Campelo et al., 2017; Hedberg et al., 2014). The adsorbed protein would impede the contact between materials and body fluids and would provide good corrosion protection (Krivosheeva et al., 2013). Thus, the materials would interact with the cells through the adsorbed protein layer and newly formed bone would accumulate on the materials (Abdal-Hay et al., 2017; Santana-Melo et al., 2017), which would further reduce the material degradation. The degradation process at in-vivo circumstance makes the materials to obtain better corrosion resistance and cytocompatibility. The serum magnesium ion levels maintained within a normal physiological range after implantation, which also indicated that the implants exhibited better biocompatibility in vivo.
5 Conclusions
This study systematically studied the property of degradation and biocompatibility of the oxidic coatings on magnesium in vitro and in vivo. In this study, we found that the in-vivo circumstance would have considerable impacts on the degradation and bone integration process. The oxidic coating could protect magnesium from fast corrosion to some extent, but the outcomes in vitro were not satisfying. When it comes to the in-vivo circumstance, the MAO-treated implants experienced little degradation and could effectively promote new bone formation. The interaction between implants and in-vivo environment would play an important role in this phenomenon, and the specific mechanism should be further investigated.
Conflict of interest
The authors declare no competing financial interest.
Acknowledgment
This work was jointly supported by the National Natural Science Foundation of China (31370962 and 31670980), the Shanghai Committee of Science and Technology (17441904000), Shanghai Rising-Star Program (15QA1404100), as well as the Youth Innovation Promotion Association CAS (2015204).
References
- Fabrication of highly porous biodegradable biomimetic nanocomposite as advanced bone tissue scaffold. Arab. J. Chem.. 2017;10:240-252.
- [Google Scholar]
- Sulfonated chitosan and dopamine based coatings for metallic implants in contact with blood. Mater. Sci. Eng. C Mater. Biol. Appl.. 2017;72:682-691.
- [Google Scholar]
- In vivo study of magnesium plate and screw degradation and bone fracture healing. Acta Biomater.. 2015;18:262-269.
- [Google Scholar]
- Dual ions implantation of zirconium and nitrogen into magnesium alloys for enhanced corrosion resistance, antimicrobial activity and biocompatibility. Colloids Surf. B, Biointerfaces. 2016;148:200-210.
- [Google Scholar]
- Cytocompatibility and early inflammatory response of human endothelial cells in direct culture with Mg-Zn-Sr alloys. Acta Biomater.. 2017;48:499-520.
- [Google Scholar]
- Preparation and characterization of a calcium-phosphate-silicon coating on a Mg-Zn-Ca alloy via two-step micro-arc oxidation. Phys. Chem. Chem. Phys. : PCCP. 2017;19:15110-15119.
- [Google Scholar]
- The potential of isotopically enriched magnesium to study bone implant degradation in vivo. Acta Biomater.. 2017;51:526-536.
- [Google Scholar]
- Cytotoxicity assessment of adipose-derived mesenchymal stem cells on synthesized biodegradable Mg-Zn-Ca alloys. Mater. Sci. Eng. C Mater. Biol. Appl.. 2016;69:584-597.
- [Google Scholar]
- Microstructure and degradation performance of biodegradable Mg-Si-Sr implant alloys. Mater. Sci. Eng. C Mater. Biol. Appl.. 2017;71:25-34.
- [Google Scholar]
- Correlation between surface physicochemical properties and the release of iron from stainless steel AISI 304 in biological media. Colloids Surf. B, Biointerfaces. 2014;122:216-222.
- [Google Scholar]
- Synthesis and modeling of uniform complex metal oxides by close-proximity atmospheric pressure chemical vapor deposition. ACS Appl. Mater. Interfaces. 2015;7:10684-10694.
- [Google Scholar]
- Novel biodegradable calcium phosphate/polymer composite coating with adjustable mechanical properties formed by hydrothermal process for corrosion protection of magnesium substrate. J. Biomed. Mater. Res. Part B, Appl. Biomater.. 2016;104:1643-1657.
- [Google Scholar]
- Electrochemical characterization and transport phenomena of polystyrene based barium-magnesium phosphate composite membrane. Arab. J. Chem.. 2016;9:S699-S706.
- [Google Scholar]
- Effect of water-soluble silicon supplementation on bone status and balance of calcium and magnesium in male mice. Biolog. Trace Element Res.. 2014;158:238-242.
- [Google Scholar]
- Nanostructured composite layers of mussel adhesive protein and ceria nanoparticles. Langmuir ACS J. Surf. Colloids. 2013;29:9551-9561.
- [Google Scholar]
- Magnesium with micro-arc oxidation coating and polymeric membrane: an in vitro study on microenvironment. J. Mater. Sci. Mater. Med.. 2015;26:147.
- [Google Scholar]
- Enhanced in vitro and in vivo performance of Mg-Zn-Y-Nd alloy achieved with APTES pretreatment for drug-eluting vascular stent application. ACS Appl. Mater. Interfaces. 2016;8:17842-17858.
- [Google Scholar]
- Evaluation of magnesium ions release, biocorrosion, and hemocompatibility of MAO/PLLA-modified magnesium alloy WE42. J. Biomed. Mater. Res. Part B, Appl. Biomater.. 2011;96:101-109.
- [Google Scholar]
- Surface modification and property analysis of biomedical polymers used for tissue engineering. Colloids Surfaces B, Biointerfaces. 2007;60:137-157.
- [Google Scholar]
- Chondrogenic differentiation of ATDC5-cells under the influence of Mg and Mg alloy degradation. Mater. Sci. Eng. C Mater. Biol. Appl.. 2017;72:378-388.
- [Google Scholar]
- Cytocompatibility of magnesium and AZ31 alloy with three types of cell lines using a direct in vitro method. J. Mater. Sci. Mater. Med.. 2016;27:145.
- [Google Scholar]
- Use of quantile regression to determine the impact on total health care costs of surgical site infections following common ambulatory procedures. Ann. Surgery. 2017;265:331-339.
- [Google Scholar]
- Enhanced corrosion resistance and biocompatibility of magnesium alloy by Mg-Al-layered double hydroxide. ACS Appl. Mater. Interfaces. 2016;8:35033-35044.
- [Google Scholar]
- Enhanced antibacterial properties, biocompatibility, and corrosion resistance of degradable Mg-Nd-Zn-Zr alloy. Biomaterials. 2015;53:211-220.
- [Google Scholar]
- In vitro study of nanostructured diopside coating on Mg alloy orthopedic implants. Mater. Sci. Eng. C Mater. Biol. Appl.. 2014;41:168-177.
- [Google Scholar]
- In vivo assessments of bioabsorbable AZ91 magnesium implants coated with nanostructured fluoridated hydroxyapatite by MAO/EPD technique for biomedical applications. Mater. Sci. Eng. C Mater. Biol. Appl.. 2015;48:21-27.
- [Google Scholar]
- Electrospun ultrathin PBAT/nHAp fibers influenced the in vitro and in vivo osteogenesis and improved the mechanical properties of neoformed bone. Colloids Surf. B, Biointerfaces. 2017;155:544-552.
- [Google Scholar]
- Long-term osseointegration of 3D printed CoCr constructs with an interconnected open-pore architecture prepared by electron beam melting. Acta Biomater.. 2016;36:296-309.
- [Google Scholar]
- Comparison study of different coatings on degradation performance and cell response of Mg-Sr alloy. Mater. Sci. Eng. C Mater. Biol. Appl.. 2016;69:95-107.
- [Google Scholar]
- The role of oxidative stress in pro-inflammatory activation of human endothelial cells on Ti6Al4V alloy. Biomaterials. 2013;34:8075-8085.
- [Google Scholar]
- MnTe semiconductor-sensitized boron-doped TiO2 and ZnO photoelectrodes for solar cell applications. J. Colloid Interface Sci.. 2013;405:78-84.
- [Google Scholar]
- Biodegradable magnesium (Mg) implantation does not impose related metabolic disorders in rats with chronic renal failure. Sci. Rep.. 2016;6:26341.
- [Google Scholar]
- In vitro degradation, hemolysis, and cytocompatibility of PEO/PLLA composite coating on biodegradable AZ31 alloy. J. Biomed. Mater. Res. Part B, Appl. Biomater.. 2015;103:342-354.
- [Google Scholar]
- Biomechanical stability of novel mechanically adapted open-porous titanium scaffolds in metatarsal bone defects of sheep. Biomaterials. 2015;46:35-47.
- [Google Scholar]
- The effect of an acute and 7-day administration of magnesium chloride on magnesium concentration in the serum, erythrocytes, and brain of rats. Pharmacol. Rep. : PR.. 2016;68:289-291.
- [Google Scholar]
- In vivo stimulation of bone formation by aluminum and oxygen plasma surface-modified magnesium implants. Biomaterials. 2013;34:9863-9876.
- [Google Scholar]
- Adjustment of the ratio of Ca/P in the ceramic coating on Mg alloy by plasma electrolytic oxidation. Appl. Surf. Sci.. 2009;255:6724-6728.
- [Google Scholar]
- Surface modification of biodegradable porous Mg bone scaffold using polycaprolactone/bioactive glass composite. Mater. Sci. Eng. C Mater. Biol. Appl.. 2015;49:436-444.
- [Google Scholar]
- Ag-Incorporated FHA coating on pure Mg: degradation and in vitro antibacterial properties. ACS Appl. Mater. Interfaces. 2016;8:5093-5103.
- [Google Scholar]
- Vascularized bone grafting fixed by biodegradable magnesium screw for treating osteonecrosis of the femoral head. Biomaterials. 2016;81:84-92.
- [Google Scholar]